Matrix metalloproteinase-13 and interleukin-6: A novel diagnostic and therapeutic target for early detection and management of osteoarthritis

INTRODUCTION

Osteoarthritis (OA) is a collection of mechanical disorders that primarily affect joints, encompassing the gradual deterioration of key components such as articular cartilage and subchondral bone.^1,2 This condition is most frequently encountered among middle-aged and elderly individuals. Factors such as aging and obesity have been strongly linked to the onset and progression of OA.^3-5 Moreover, various other factors, including gender, race, ethnicity, genetics, dietary habits, smoking, diabetes, and joint injuries or trauma, contribute to the development of OA.⁶

OA is the second most common rheumatological disorder in India, with prevalence ranging from 22% to 39%.⁷ The prevalence is higher in females (25.39%) as compared to males (19.3%), and people > 60 years are at major risk for OA. Knee OA is most prevalent (16.5%), followed by hip OA (5.1%).⁴ OA has the potential to impact any synovial joint, yet it most frequently affects the hip, knee, hand, foot, and spine. The manifestations of OA often develop gradually and can include joint pain, restricted range of motion, stiffness following periods of inactivity, soreness, the sensation of crepitus (a crackling or grinding sound), and joint hypertrophy (enlargement of the joint). In the chronic stages of OA, one may observe localized disintegration and softening of the articular cartilage as significant pathological changes.⁸

The current diagnostic approach for OA relies on both radiological and clinical assessments. Radiography, while valuable for tracking disease progression, has limitations due to its low sensitivity, making it less suitable for early diagnosis.⁹ For the diagnosis and grading of OA, the Kellgren and Lawrence (KL) criteria stand as the most widely accepted standards, also endorsed by the World Health Organization for epidemiological investigations of OA.^10,11 Additionally, the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score serves as a valuable tool for evaluating pain in OA patients. This assessment method encompasses a comprehensive evaluation of pain, stiffness, and functionality.¹²

For the early diagnosis and monitoring of OA, there is a need to investigate and validate new options, such as biochemical & proteomic markers. In OA, the extracellular matrix (ECM) is degraded within synovial joints, particularly those in the knee. The injury in the synovial joints leads to the activation of inflammatory cascades and produces several inflammatory mediators such as Tumor necrosis factor alpha and interleukin-1, which heighten inflammation and augment the synthesis of matrix metalloproteinase (MMP) enzymes, further worsening the pathogenesis of OA.¹³ MMP-13 belongs to the family of MMPs; it is a zinc-dependent peptidase originating from chondrocytes seen in the cartilage. Reports suggest that MMP-13 has a role in the degradation of proteoglycan molecules.¹⁴ MMP overexpression (e.g., MMP-9 and MMP-13) is thought to be important in the development of OA.¹⁵ IL-6 is a cytokine that promotes inflammation and is involved in the pathogenesis of OA.¹⁶ The relationship between MMP-13 and interleukin-6 (IL-6) and the severity of OA in the Indian population is poorly observed. To establish their relationship with disease development and severity, it is important to investigate the levels of MMP13 and IL6 in OA patients. This study helps to improve our understanding of the disease and may lead to the development of new diagnostic & therapeutic targets.

MATERIAL AND METHODS

In this cross-sectional study, a total of 225 study subjects were enrolled for eligibility from the outpatient Department of Orthopaedics, Hakeem Abdul Hameed Centenary Hospital (HACH), New Delhi. The study subjects with the following conditions were included in the study: i) Both males and females aged >40 years. ii) Radiological/clinically confirmed knee OA patients. iii) Highly sensitive C-reactive protein (hs-CRP) level <10 mg/L. Further, subjects age>40 years without any clinical symptoms and comorbid conditions were recruited in the healthy control group. A total of 225 study subjects were screened for eligibility, and 113 subjects were not fit for the study as per the inclusion criteria. Finally, 112 subjects in both control (n=56) and test groups (n=56) were included in this study for analysis. We calculated the sample size using the variables α=5% (significance level), β=90% (probability of detecting a significant result), μd=658, Z= 95% confidence interval, and calculated 56 subjects in each group. The study was approved by the institutional ethics committee (EC/NEW/INST/2020/961).

Methodology

In present study, the severity of OA was assessed using the KL grading system, which evaluates osteophyte formation, subchondral sclerosis, and subchondral cysts through radiographic examination. KL grading is represented on a scale from 0 to 4, with higher scores indicating more advanced OA [Figure 1]. The WOMAC Osteoarthritis Index evaluates pain, stiffness, and function and is considered the most accepted indicator for the assessment of OA. It assesses three key dimensions: pain, stiffness, and function. Specifically, it includes 17 questions related to function, two questions regarding stiffness, and five questions concerning pain.¹⁷

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Figure 1:

On an anterior-posterior view of a radiograph taken while standing, Kallgren and Lawrence created a radiological scale to categorise knee OA into four categories. “Grade 0 is no radiographic evidence of osteoarthritis, Grade 1 is defined as minute osteophytes of doubtful clinical significance (white arrow), Grade 2 is definite osteophytes with unimpaired joint space (white arrow), Grade 3 is definite osteophytes with moderate joint space narrowing (white arrow), Grade 4 is definite osteophytes with severe joint space narrowing and subchondral sclerosis (white arrow sing).”

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RESULTS

MMP-13 and IL-6 levels in control and osteoarthritis study subjects

The estimated serum IL-6 mean values were 118.7±7.81 (pg/ml) and 41.35 ± 3.43 (pg/ml) in OA and control group, respectively whose difference was statistically significant. Besides, IL-6 mean levels in urine samples of OA and control subjects were 84.24 ± 6.31 (pg/ml) and 26.00 ± 4.42 (pg/ml), respectively and the difference was further statistically significant (p<0.001. The level of MMP-13 in OA and control group was 63.77 ± 3.59 (µg/l) and 32.82 ± 2.59 (µg/l), respectively. The difference of MMP-13 values in both groups were statistically significant (p<0.001) [Figure 2].

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Figure 2:

MMP-13 and IL-6 levels in control (n = 56) and osteoarthritis (n = 56) study subjects. Data are expressed as mean ± SEM, p‐value is calculated using unpaired “t” test. The difference among all these groups is statistically significant (p <0 .05). MMP- 13: Matrix metalloproteinase- 13, IL-6: Interleukin-6.

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DISCUSSION

The incidence of OA has seen a significant, exponential increase in recent years. Currently, the diagnosis of OA relies on clinical and radiographic criteria that fail to detect early symptoms of OA and its progression until the condition becomes severe.⁹ The present study suggests that IL-6 and MMP-13 are potential biomarkers in the early prognosis of OA. The significantly higher BMI in OA subjects in comparison to the control group [Table 1] indicates that the obese individuals may have a higher risk of developing OA. Our findings in this regard are supported by previous work by Conrozier et al., (1998), who showed that BMI and obesity are correlated with inflammation, as well as the progression of OA.¹⁸ In the present study, we observed that aliquot varus was significantly increased in OA patients as compared to the control group, and this observation was also supported by Sharma et al., (2010).¹⁹ Hanada et al., (2016) observed that ESR and hs-CRP were higher in patients with knee OA, and a similar observation was also found in the present study.²⁰ The possible reason for this elevated hs-CRP may be attributed to its role in driving a specific inflammatory pathway that is associated with osteophyte development. Further, CRP could be involved in lipid-driven local cartilage changes via OA-induced upregulation and activation of phospholipase A2 (PLA2) enzymes by chondrocytes. Both ESR and CRP are commonly used as markers of the acute-phase inflammation response. The present study revealed that serum uric acid levels were increasing in OA patients. Interestingly, Denoble et al. (2011) also found the elevated levels of serum uric acid (>6 mg/dL) in knee OA patients and postulated that it is due to activation of NLRP3 inflammasome, which triggers the release of inflammatory markers.²¹ Further, Li et al., (2017), established the inverse relationship between serum calcium concentration and radiographic proven severity of OA in the patients.¹⁶

The radiographic severity in OA is often assessed by KL grade, which ranges from grade I to grade IV. The present study analyzed the association of MMP-13, serum IL-6, urinary IL-6, and other biochemical parameters (hs-CRP, ESR, and serum uric acid) with KL grade in OA subjects. There was a statistically significant association between MMP-13 levels and KL grading, which indicates that MMP-13 could be used for earlier diagnostic purposes. MMP13 is a family of functionally and structurally related zinc endopeptidases that cleave proteins of the ECM, including collagens, elastin, matrix glycoproteins, and proteoglycans, and are responsible for the degeneration of articular cartilage.²² Furthermore, levels of serum IL-6 were also significantly elevated in subjects with OA as compared to control subjects. The difference in IL-6 in both groups was statistically significant (p<0.05). Imamura et al., (2015), also described a similar observation^23, and therefore it is a potential biomarker for the assessment of OA severity. IL-6 is a proinflammatory cytokine that activates receptors: membrane-bound IL-6 receptor (IL6R) and soluble IL-6 receptor (sIL-6R).²⁴ The generation of IL-6 by chondrocytes is quite low in physiological conditions.

There are reports that angiogenesis and chronic inflammation are the two processes that are intertwined in OA patients. The role of nucleoprotein and inflammatory markers in the pathogenesis of knee OA has been elucidated in Figure 3. Macrophages and mast cells, which are abundant in chronically inflamed osteoarthritic synovium, are responsible for the inflammatory cascade. It is postulated that mechanical stress, aging, obesity, and other factors stimulate the vascular endothelial growth factor, which leads to an increase in osteoblast activity in the knee.²⁵ Further, it secretes pro-inflammatory cytokines that trigger fibroblasts to release inflammatory cytokines. The inflammatory cascade increases the chondrocyte expression of matrix proteins such as MMP-13, causing cartilage breakdown and bone remodeling. Sample size and follow-up of the study subjects for variations in biochemical markers are the limitations of the present study.

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Figure 3:

Putative biochemical elucidation of inflammation in osteoarthritis and its further role in pathophysiology. VEFG: Vascular endothelial growth factor, PPR: Pro-renin receptor, PPR : Pattern recognition receptor, TLR 4/2: Toll-like receptors 4 and 2, TNFα: Tumor necrosis factor alpha, MMP- 13: Matrix metalloproteinase- 13, IL-6: Interleukin-6.

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CONCLUSION

The present study revealed that MMP-13 and serum/urinary IL-6 levels were significantly increased in OA patients when compared with the control group. The MMP-13 values positively correlated with the KL grading of OA. Further, MMP-13 and serum IL-6 significantly correlated with the WOMAC score. In conclusion, assessment of MMP-13 and serum IL-6 in OA patients may be considered as a diagnostic and therapeutic target for early detection and management of OA. The MMP-13 and serum IL-6 may be effectively utilized for the early detection of OA, which would largely benefit the affected individuals through timely treatment and precautions.